A retinoic acid responsive Hoxa3 transgene expressed in embryonic pharyngeal endoderm, cardiac neural crest and a subdomain of the second heart field.
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SUBMITTER: Diman NY
PROVIDER: S-EPMC3217993 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:ObjectivesNormal commitment of the endoderm of the third pharyngeal pouch (3PP) is essential for the development and differentiation of the thymus. The aim of this study was to investigate the role of transcription factor HOXA3 in the development and differentiation of 3PP endoderm (3PPE) from human embryonic stem cells (hESCs).MethodsThe 3PPE was differentiated from hESC-derived definitive endoderm (DE) by mimicking developmental queues with Activin A, WNT3A, retinoic acid and BMP4. The function of 3PPE was assessed by further differentiating into functional thymic epithelial cells (TECs). The effect of HOXA3 inhibition on cells of 3PPE was subsequently investigated.ResultsA highly efficient approach for differentiating 3PPE cells was developed and these cells expressed 3PPE related genes HOXA3, SIX1, PAX9 as well as EpCAM. 3PPE cells had a strong potential to develop into TECs which expressed both cortical TEC markers K8 and CD205, and medullary TEC markers K5 and AIRE, and also promoted the development and maturation of T cells. More importantly, transcription factor HOXA3 not only regulated the differentiation of 3PPE, but also had a crucial role for the proliferation and migration of 3PPE cells. Our further investigation revealed that HOXA3 controlled the commitment and function of 3PPE through the regulation of Wnt signaling pathway by activating EPHB2.ConclusionOur results demonstrated that HOXA3 functioned as the on-off switch to regulate the development of hESC-derived 3PPE through EPHB2-mediated Wnt pathway, and our findings will provide new insights into studying the development of 3PP and thymic organ in vitro and in vivo.
| S-EPMC10800530 | biostudies-literature
Project description:Coordinated transcriptional and epigenetic mechanisms that direct development of the later differentiating second heart field (SHF) progenitors remain largely unknown. Here, we show that a novel zebrafish histone deacetylase 1 (hdac1) mutant allele cardiac really gone (crg) has a deficit of ventricular cardiomyocytes (VCs) and smooth muscle within the outflow tract (OFT) due to both cell and non-cell autonomous loss in SHF progenitor proliferation. Cyp26-deficient embryos, which have increased retinoic acid (RA) levels, have similar defects in SHF-derived OFT development. We found that nkx2.5+ progenitors from Hdac1 and Cyp26-deficient embryos have ectopic expression of ripply3, a transcriptional co-repressor of T-box transcription factors that is normally restricted to the posterior pharyngeal endoderm. Furthermore, the ripply3 expression domain is expanded anteriorly into the posterior nkx2.5+ progenitor domain in crg mutants. Importantly, excess ripply3 is sufficient to repress VC development, while genetic depletion of Ripply3 and Tbx1 in crg mutants can partially restore VC number. We find that the epigenetic signature at RA response elements (RAREs) that can associate with Hdac1 and RA receptors (RARs) becomes indicative of transcriptional activation in crg mutants. Our study highlights that transcriptional repression via the epigenetic regulator Hdac1 facilitates OFT development through directly preventing expression of the RA-responsive gene ripply3 within SHF progenitors.
| S-EPMC6538190 | biostudies-literature
Project description:Normal commitment of the endoderm of the third pharyngeal pouch (3PP) is essential for the development and differentiation of the thymus. The aim of this study was to investigate the role of transcription factor HOXA3 in the development and differentiation of the third pharyngeal pouch endoderm from human embryonic stem cells (hESCs). 3PP endoderm (3PPE) was differentiated from hESC-derived definitive endoderm (DE) by mimicking developmental queues with Activin A, WNT3A, retinoic acid and BMP4. The function of 3PPE was assessed by further differentiating into functional thymic epithelial cells. The effect of HOXA3 inhibition on cells of 3PPE was subsequently investigated. A highly efficient approach for differentiating 3PPE cells has been developed and these cells expressed 3PPE related genes HOXA3, SIX1, PAX9 as well as EpCAM. 3PPE cells had a strong potential to develop into thymic epithelia which expressed both the cortical epithelial cell markers K8 and CD205, and the medullary epithelial cell markers K5 and AIRE, and also promoted T cell maturation. More importantly, transcription factor HOXA3 not only regulated the differentiation of 3PPE, but also had a crucial role for the proliferation and migration 3PPE cells. Our further investigation revealed that HOXA3 controlled the commitment and function of 3PPE through the regulation of the Wnt signaling pathway by EPHB2. Our results demonstrated that HOXA3 functioned as the on-off switch to regulate the development of hESC-derived 3PPE through EPHB2-mediated Wnt pathway, and our findings will provide new insights into studying the development of the third pharyngeal pouch and thymic organ in vitro and in vivo.
2023-07-24 | GSE237751 | GEO
Project description:Developmental defects affecting the heart and aortic arch arteries are a significant phenotype observed in individuals with 22q11 deletion syndrome and are caused by a microdeletion on chromosome 22q11. TBX1, one of the deleted genes, is expressed throughout the pharyngeal arches and is considered a key gene, when mutated, for the arch artery defects. Pax9 is expressed in the pharyngeal endoderm and is downregulated in Tbx1 mutant mice. We show here that Pax9-deficient mice are born with complex cardiovascular malformations that affect the outflow tract and aortic arch arteries with failure of the 3rd and 4th pharyngeal arch arteries to form correctly. Transcriptome analysis indicated that Pax9 and Tbx1 may function together, and mice double heterozygous for Tbx1/Pax9 presented with a significantly increased incidence of interrupted aortic arch when compared with Tbx1 heterozygous mice. Using a novel Pax9Cre allele, we demonstrated that the site of this Tbx1-Pax9 genetic interaction is the pharyngeal endoderm, therefore revealing that a Tbx1-Pax9-controlled signalling mechanism emanating from the pharyngeal endoderm is required for crucial tissue interactions during normal morphogenesis of the pharyngeal arch artery system.
| S-EPMC6765178 | biostudies-literature
Project description:Coordinated transcriptional and epigenetic mechanisms that direct development of the later differentiating second heart field (SHF) progenitors remain largely unknown. Here, we show that a novel zebrafish histone deacetylase 1 (hdac1) mutant allele cardiac really gone (crg) has a deficit of ventricular CMs and smooth muscle within the outflow tract (OFT) due to both cell and non-cell autonomous loss in SHF progenitor proliferation. Cyp26-deficient embryos, which have increased retinoic acid (RA) levels, have similar defects in SHF-derived OFT development. We found that nkx2.5+ progenitors from hdac1 and cyp26-deficient embryos have ectopic expression of ripply3, a transcriptional co-repressor of T-box transcription factors that is normally restricted to the posterior pharyngeal endoderm. Furthermore, the ripply3 expression domain is expanded anteriorly into the posterior nkx2.5+ progenitor domain in crg mutants. Importantly, excess ripply3 is sufficient to repress VC development, while genetic depletion of Ripply3 and Tbx1 in crg mutants can partially restore VC number. We find that in crg mutants the epigenetic signature at RA response elements (RAREs) that can associate with Hdac1 and RA receptors (RARs) becomes indicative of transcriptional activation. Our study highlights that transcriptional repression via the epigenetic regulator Hdac1 facilitates OFT development through directly preventing expression of the RA-responsive gene ripply3 within SHF progenitors.
2019-05-11 | GSE126747 | GEO
Project description:In vertebrates, face and throat structures, such as jaw, hyoid and thyroid cartilages develop from a rostrocaudal metameric series of pharyngeal arches, colonized by cranial neural crest cells (NCCs). Colinear Hox gene expression patterns underlie arch specific morphologies, with the exception of the first (mandibular) arch, which is devoid of any Hox gene activity. We have previously shown that the first and second (hyoid) arches share a common, Hox-free, patterning program. However, whether or not more posterior pharyngeal arch neural crest derivatives are also patterned on the top of the same ground-state remained an unanswered question. Here, we show that the simultaneous inactivation of all Hoxa cluster genes in NCCs leads to multiple jaw and first arch-like structures, partially replacing second, third and fourth arch derivatives, suggesting that rostral and caudal arches share the same mandibular arch-like ground patterning program. The additional inactivation of the Hoxd cluster did not significantly enhance such a homeotic phenotype, thus indicating a preponderant role of Hoxa genes in patterning skeletogenic NCCs. Moreover, we found that Hoxa2 and Hoxa3 act synergistically to pattern third and fourth arch derivatives. These results provide insights into how facial and throat structures are assembled during development, and have implications for the evolution of the pharyngeal region of the vertebrate head.
| S-EPMC4482666 | biostudies-literature
Project description:Branching morphogenesis depends on the precise temporal and spatial control of epithelial dynamics. In the vertebrate head, endodermal branches, called pharyngeal pouches, form through the transient stratification, collective migration and reorganization of epithelial cells into bilayers. Here, we report novel requirements for the EphrinB ligands B2a and B3b, the Ephb4a receptor and the Pak2a kinase in the development of pouches and the posterior facial skeleton that depends on pouches for its segmentation. Time-lapse imaging in zebrafish shows that EphB-Pak2a signaling is required to stabilize pouch epithelial cells at the end of branching morphogenesis. Transgenic rescue experiments further demonstrate that endodermal Eph-ephrin signaling promotes pouch integrity by targeting Pak2a to the plasma membrane, where subsequent activation by Wnt4a-Cdc42 signaling increases junctional E-cadherin in maturing pouches. Integration of Eph-ephrin and Wnt4a signaling through Pak2a thus signals the end of branching morphogenesis by increasing intercellular adhesion that blocks further epithelial rearrangements.
| S-EPMC4360181 | biostudies-literature
Project description:Neurogenin3 is a member of the basic helix-loop-helix ('bHLH') family of transcription factors. It plays a crucial role in the commitment of embryonic endoderm into the pancreatic differentiation programme. This factor is considered to act upstream of a cascade of other transcription factors, leading to the fully differentiated endocrine phenotype. Direct observation of the sequential activation of these factors starting from Neurogenin3 had never been demonstrated. By using retinoic acid-derived-endoderm F9 cells as a model, the present study indicates that the ectopic expression of Neurogenin3 is able to start the differentiation pathway of endocrine pancreas. Neurogenin3 triggers the expression of several pancreatic transcription factors following a well defined temporal activation sequence. By reverse transcriptase PCR, immunohistochemistry and RIA, it is shown that stable transfected cells are able to form embryod bodies that produce insulin in response to glucose stimulation. This is the first report of a differentiation event induced by the ectopic expression of a transcription factor in embryonic pluripotent stem cells.
| S-EPMC1223320 | biostudies-other
Project description:The mechanism of action of retinoic acid (RA) is of broad relevance to cell and developmental biology, nutrition, and cancer chemotherapy. RA is known to induce expression of the Burkitt's lymphoma receptor 1 (BLR1) gene which propels RA-induced cell cycle arrest and differentiation of HL-60 human myeloblastic leukemia cells, motivating the present analysis of transcriptional regulation of blr1 expression by RA. The RA-treated HL-60 cells used here expressed all RA receptor (RAR) and retinoid X receptor (RXR) subtypes (as detected by Northern analysis) except RXRgamma. Treatment with RAR- and RXR-selective ligands showed that RARalpha synergized with RXRalpha to transcriptionally activate blr1 expression. A 5'-flanking region capable of supporting RA-induced blr1 activation in HL-60 cells was found to contain a 205-bp sequence in the distal portion that was necessary for transcriptional activation by RA. Within this sequence DNase I footprinting revealed that RA induced binding of a nuclear protein complex to an element containing two GT boxes. Electromobility shift assays (EMSAs) and supershift assays showed that this element bound recombinant RARalpha and RXRalpha. Without RA there was neither complex binding nor transcriptional activation. Both GT boxes were needed for binding the complex, and mutation of either GT box caused the loss of transcriptional activation by RA. The ability of this cis-acting RAR-RXR binding element to activate transcription in response to RA also depended on downstream sequences where an octamer transcription factor 1 (Oct1) site and a nuclear factor of activated T cells (NFATc) site between this element and the transcriptional start, as well as a cyclic AMP response element binding factor (CREB) site between the transcriptional start and first exon of the blr1 gene, were necessary. Each of these sites bound its corresponding transcription factor. A transcription factor-transcription factor binding array analysis of nuclear lysate from RA-treated cells indicated several prominent RARalpha binding partners; among these, Oct1, NFATc3, and CREB2 were identified by competition EMSA and supershift and chromatin immunoprecipitation assays as components of the complex. RA upregulated expression of these three factors. In sum the results of the present study indicate that RA-induced expression of blr1 expression depends on a novel RA response element. This cis-acting element approximately 1 kb upstream of the transcriptional start consists of two GT boxes that bind RAR and RXR in a nuclear protein complex that also contains Oct1, NFATc3, and CREB2 bound to their cognate downstream consensus binding sites.
| S-EPMC355834 | biostudies-literature
Project description:BackgroundThe T-box transcription factor Tbx1, is essential for the normal development of multiple organ systems in the embryo. One of the most striking phenotypes in Tbx1-/- embryos is the failure of the caudal pharyngeal pouches to evaginate from the foregut endoderm. Despite considerable interest in the role of Tbx1 in development, the mechanisms whereby Tbx1 controls caudal pouch formation have remained elusive. In particular, the question as to how Tbx1 expression in the pharyngeal endoderm regulates pharyngeal pouch morphogenesis in the mouse embryo is not known.ResultsTo address this question, we produced mouse embryos in which Tbx1 was specifically deleted from the pharyngeal endoderm and, as expected, embryos failed to form caudal pharyngeal pouches. To determine the molecular mechanism, we examined expression of Fgf3 and Fgf8 ligands and downstream effectors. Although Fgf8 expression is greatly reduced in Tbx1-deficient endoderm, FGF signaling levels are unaffected. Furthermore, pouch morphogenesis is only partially perturbed by the loss of both Fgf3 and Fgf8 from the endoderm, indicating that neither are required for pouch formation.ConclusionsTbx1 deletion from the pharyngeal endoderm is sufficient to cause caudal pharyngeal arch segmentation defects by FGF-independent effectors that remain to be identified.
| S-EPMC4349409 | biostudies-literature